ABSTRACT Multiple sclerosis (MS) is a multifaceted neurological disease and one of the most common causes of disability in young adults. The major hallmark of MS is an uncontrolled immune response that drives demyelination, resulting in continuously worsening cognitive impairments, and eventually leading to death. Upon clinical diagnosis of MS, numerous clinical evaluations and MR Imaging (MRI) sessions are required to assess symptoms and/or presence of lesions. Even then, prediction of outcome and choice of treatment remain a challenge for each patient. Given the role of inflammation in MS, an imaging method that could detect immune response could improve patient management and subsequent individualized therapeutic approaches. In MS lesions, mononuclear phagocytes (MPs, macrophages/microglia) are the most abundant immune cells, and drive demyelination and cell death. Interestingly, to sustain high proliferation rates, these MPs have switched from quiescent to pro-inflammatory (M1-polarized) activated state and show increased lactate production linked to increased pyruvate dehydrogenase kinase 1 (PDK1). During remissions or in response to therapies, activated MPs switch to a neuroprotective (M2) phenotype and participate in remyelination. Remarkably, M2 MPs present the unique metabolic feature of excreting high levels of arginase, an enzyme that inhibits T cells function through depletion of the arginine pool. The goal of this study is to test the hypothesis that MPs activation and M1/M2 status can be detected in MS lesions in vivo using hyperpolarized 13C MR Spectroscopic Imaging (HP 13C MRSI) and that such metabolic imaging can improve evaluation of MS progression and treatment response. Our Aims are: Aim 1. Validate 13C MRSI of HP pyruvate as an imaging method to monitor activated MPs in MS lesions in vivo. 13C MRSI of HP [1-13C] pyruvate, the most established and clinically translatable probe, will be used to detect PDK1+/activated MPs through increased HP lactate production in two well-characterized preclinical MS models. Comparison with established MRI methods will be performed to assess the sensitivity, specificity and diagnostic accuracy of the method, and its improved potential to monitor disease progression. Aim 2. Detect neuroprotective M2 MPs in MS lesions using in vivo 13C MRSI of hyperpolarized arginine. We will optimize 13C MRSI of HP guanidino-13C-arginine, the substrate of arginase, to non-invasively and specifically detect arginase+/M2 MPs in vivo through detection of HP urea in preclinical MS models. Comparison to MRI/S will also be performed to assess the improved diagnostic accuracy of this approach. Aim 3. Evaluate in vivo MR metabolic imaging to monitor response to therapies. We will combine 13C MRSI of HP [1-13C] pyruvate and HP guanidino-13C-arginine and use this multiprobe metabolic imaging approach to monitor response to clinically relevant therapies in two preclinical MS models. Comparison to MRI/S methods will be conducted to assess the improved monitoring accuracy of each method.